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Biochimica et Biophysica Acta 1587 (2002) 258–275 www.bba-direct.com Review New developments in anti-HIV chemotherapy $

Erik De Clercq*

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Received 24 January 2002; accepted 24 January 2002

Abstract

Virtually all the compounds that are currently used, or are subject of advanced clinical trials, for the treatment of human immunodeficiency virus (HIV) , belong to one of the following classes: (i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs): i.e. (AZT), (ddI), (ddC), (d4T), (3TC), (ABC), [( À )FTC], fumarate; (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs): i.e. , , , ; and (iii) protease inhibitors (PIs): i.e. , , , , and . In addition to the reverse transcriptase (RT) and protease reaction, various other events in the HIV replicative cycle can be considered as potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulfates, polysulfonates, polycarboxylates, polyoxometalates, polynucleotides, and negatively charged albumins); (ii) viral entry, through blockade of the viral coreceptors CXCR4 [bicyclam (AMD3100) derivatives] and CCR5 (TAK-779 derivatives); (iii) virus–cell fusion, through binding to the viral envelope glycoprotein (T-20, T-1249); (iv) viral assembly and disassembly, through NCp7 zinc finger-targeted agents [2,2V- dithiobisbenzamides (DIBAs), azadicarbonamide (ADA)]; (v) proviral DNA integration, through integrase inhibitors such as 4-aryl-2,4- dioxobutanoic acid derivatives; (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (flavopiridol, fluoroquinolones). Also, various new NRTIs, NNRTIs and PIs have been developed that possess, respectively: (i) improved metabolic characteristics (i.e. phosphoramidate and cyclosaligenyl pronucleotides by-passing the first phosphorylation step of the NRTIs), (ii) increased activity [‘‘second’’ or ‘‘third’’ generation NNRTIs (i.e. TMC-125, DPC-083)] against those HIV strains that are resistant to the ‘‘first’’ generation NNRTIs, or (iii) as in the case of PIs, a different, nonpeptidic scaffold [i.e. cyclic urea (mozenavir), 4-hydroxy-2-pyrone ()]. Nonpeptidic PIs may be expected to inhibit HIV mutant strains that have become resistant to peptidomimetic PIs. Given the multitude of molecular targets with which anti-HIV agents can interact, one should be cautious in extrapolating the mode of action of these agents from cell-free enzymatic assays to intact cells. Two examples in point are L-chicoric acid and the nonapeptoid CGP64222, which were initially described as an or Tat antagonist, respectively, but later shown to primarily act as virus adsorption/entry inhibitors, the latter through blockade of CXCR4. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Human immunodeficiency virus (HIV); Reverse transcriptase (HIV); Protease (HIV); CXCR4 (HIV); CCR5 (HIV); Integrase (HIV); Fusion (HIV); Transcription (HIV)

Abbreviations: HIV, human immunodeficiency virus; NRTIs, nucleoside/nucleotide reverse transcriptase inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors; PIs, protease inhibitors; DIBA, 2,2V-dithiobisbenzamide; ADA, azadicarbonamide; AIDS, acquired immune deficiency syndrome; HSV, herpes simplex virus; STD, sexually transmitted disease; MIP-1a and -1h, macrophage inflammatory proteins; SDF-1, stromal-cell derived factor; PBMCs, peripheral blood mononuclear cells; TM4, transmembrane segment; SI, syncytium-inducing; NSI, non-syncytium-inducing; NOBA, 3-nitrosobenzamide; AZT, zidovudine; ddI, didanosine; ddC, zalcitabine; d4T, stavudine; 3TC, lamivudine; ABC, abacavir; bis(POM)-PMEA, bis(pivaloyloxymethyl)-9-(2- phosphonylmethoxyethyl)adenine, adefovir dipivoxyl; bis(POC)-PMPA, bis(isopropyloxycarbonyloxymethyl)-(R)-9-(2-phosphonylmethoxypropyl)adenine, tenofovir disoproxil; dOTC, (F)2V-deoxy-3V-oxa-4-thiocytidine; (À)FTC, emtricitabine; DAPD, , (À)-h-D-2,6-diaminopurine dioxolane; bis(SATE)ddAMP, bis(S-acetyl-2-thioethyl)phosphotriester of ddA $ Proceedings of the 8th International Symposium on Molecular Aspects of Chemotherapy, Gdansk, Poland, 5–9 September 2001. * Tel.: +32-16-337341; fax: +32-16-337340. E-mail address: [email protected] (E. De Clercq).

0925-4439/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S0925-4439(02)00089-3 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 259

1. Introduction have been formally approved, for the treatment of HIV infections, will undoubtedly improve the prognosis of Combination therapy, comprising at least three anti- patients with AIDS and AIDS-associated diseases. Here, I human immunodeficiency virus (HIV) drugs, has become will primarily address those new anti-HIV compounds that (i) the standard treatment of acquired immune deficiency have emerged as promising anti-HIV drug candidates during syndrome (AIDS) or HIV-infected patients. Virtually all the last few years, that (ii) are in preclinical or early-clinical drugs that have been licensed for clinical use (or made development, and that (iii) are targeted at well-defined steps available through expanded access programmes) for the in the HIV replicative cycle. treatment of HIV infections fall into one of the following three categories: (i) nucleoside/nucleotide reverse transcrip- tase inhibitors (NRTIs), that, following two phosphorylation 2. Virus adsorption (gp120) inhibitors steps (tenofovir) or three phosphorylation steps [zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), A great variety of polyanionic compounds have been lamivudine (3TC), abacavir (ABC)], act, as chain termina- described to block HIV replication through interference with tors, at the substrate binding site of the reverse transcriptase virus adsorption (or binding) to the cell surface: i.e. poly- (RT); (ii) non-nucleoside reverse transcriptase inhibitors sulfates, polysulfonates, polycarboxylates, polyphosphates, (NNRTIs) that interact with the RT at an allosteric, non- polyphosphonates, polyoxometalates, etc. This class of substrate binding site (nevirapine, delavirdine, efavirenz); compounds also comprises the cosalane analogues (1) con- and (iii) protease inhibitors (PIs) that specifically inhibit, as taining the polycarboxylate pharmacophore [3], as well as peptidomimetics, the virus-associated protease (saquinavir, the sulfated polysaccharides extracted from sea algae [4]. ritonavir, indinavir, nelfinavir, amprenavir, lopinavir). All these compounds, whether synthetic or of natural origin, Guidelines to the major clinical trials with these compounds are assumed to exert their anti-HIV activity by shielding off have been recently published [1]. the positively charged sites in the V3 loop of the viral Although the long-term goal of eradicating the virus from envelope glycoprotein (gp120) [5,6], which is necessary for latently and chronically infected cells remains forbidding [2], virus attachment to the cell surface heparan sulfate, a the advent of so many new compounds other than those that primary binding site, before a more specific binding occurs 260 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 to the CD4 receptor of the CD4+ cells, and to the CXCR4 ligand, namely SDF-1 (‘‘stromal-cell derived factor’’) has coreceptor of the CXCR4+ cells (the latter in the case of X4 been identified. Of these chemokines, the LD78h isoform of and dual tropic X4/R5 HIV strains). Heparan sulfate is MIP-1a has emerged as the most potent chemokine for widely expressed on animal cells and, as it is involved in inhibiting HIV-1 in peripheral blood mononuclear the virus-cell binding of a broad spectrum of enveloped cells (PBMCs) [14,15] as well as monocytes/macrophages viruses, including herpes simplex virus (HSV) [7], dengue [16]. virus [8] and other flaviviruses (i.e. Japanese encephalitis TAK-779, a quaternary ammonium derivative (2) is the virus) [9], it also explains why polysulfates have a broad- first nonpeptidic molecule that has been described to block spectrum antiviral activity against HIV, HSV and various the replication of M-tropic R5 HIV-1 strains at the CCR5 other enveloped viruses [10]. level [17].

The major role of polysulfates or polyanionic substances A binding site for TAK-779 has been identified within in general in the management of HIV infections may reside the transmembrane helices 1, 2, 3 and 7 of CCR5 [18]. in the prevention of sexual transmission of HIV infection, as TAK-779 has been found to inhibit R5 HIV-1 strains in the these compounds, if applied as a vaginal formulation, may nanomolar concentration range, while not affecting X4 successfully block HIV infection through both virus-to-cell HIV-1 strains at 10,000-fold higher concentrations [17]. and cell-to-cell contact. These compounds therefore merit TAK-779 is not a ‘‘pure’’ CCR5 antagonist, as it also being pursued as vaginal microbicides. The fact that in demonstrates some antagonism towards CCR2b. Unlike addition to their anti-HIV activity, these polyanionic sub- RANTES, TAK-779 does not induce internalization of stances, as demonstrated, for example, for poly(sodium(4- CCR5. The clinical potential of TAK-779 and its conge- styrene)sulfonate), also inhibit other sexually transmitted ners [19] in the therapy and/or prophylaxis of HIV-1 disease (STD) pathogens, i.e. HSV, Neisseria gonorrheae infections remains to be further explored. Meanwhile, and Chlamydia trachomatis [11], further adds to their several new CCR5 antagonists have been reported potential therapeutic and preventive value. [20,21], and a lead clinical candidate (SCH C) for further development has been identified. Almost simultaneously [22–24], three compounds, i.e. 3. Viral coreceptor antagonists the bicyclam AMD3100 [22], [Tyr-5,12,Lys-7]polyphemu- sin or T22 [23] and the nonapeptide (D-Arg)9 or ALX40- To enter cells, following binding with the CD4 receptor, 4C [24] were announced as CXCR4 antagonists, blocking the HIV-1 particles must interact, again through the viral the replication of T-tropic X4, but not M-tropic R5, HIV-1 envelope glycoprotein gp120, with the CXCR4 coreceptor strains through selective antagonism of CXCR4. The [12] or CCR5 coreceptor [13]. CXCR4 is the coreceptor for bicyclams are the most specific and most potent CXCR4 HIV-1 strains that infect T-cells (T-tropic or X4 strains), and antagonists that have been described to date [25,26]. The CCR5 is the coreceptor for HIV-1 strains that infect macro- bicyclams had been known as potent and selective HIV phages (M-tropic or R5 strains). CXCR4 and CCR5 have inhibitors for a number of years [27,28], before their not evolved simply to act as coreceptors for HIV entry; they target of action was identified as the CXCR4 coreceptor normally act as receptors for chemokines (chemoattractant [22,29,30]. The bicyclam AMD3100 (3) inhibits the cytokines). The normal ligands for CCR5 are RANTES replication of X4 HIV-1 strains within the nanomolar (‘‘regulated upon activation, normal T-cell expressed and concentration range [28]. As it is not toxic to the host secreted’’) and MIP-1a and -1h (‘‘macrophage inflamma- cells at concentrations up to 500 AM, its selectivity index, tory proteins’’), whereas for CXCR4, only one natural or ratio of 50% cytotoxic concentration (CC50) to 50% E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 261 antivirally effective concentration (EC50) can be estimated and TM6, may represent crucial sites of interaction with at > 100,000. the bicyclam AMD3100 [32]. When the bicyclam AMD3100 was added to PBMC infected with clinical HIV isolates displaying the syncy- tium-inducing (SI) phenotype, these strains reverted to the non-syncytium-inducing (NSI) phenotype, and, concomi- tantly, these strains switched from CXCR4 to CCR5 cor- eceptor use [33]. These findings indicate that selective blockade of CXCR4 by AMD3100 may prevent the switch from the less pathogenic M-tropic R5 to the more patho- genic T-tropic X4 strains of HIV, which in vivo heralds the progression to AIDS. AMD3100 has proved efficacious, alone and in combination with other anti-HIV drugs, in achieving a marked reduction in viral load in the SCID-hu Thy/Liv mouse model [34]. Following a phase I clinical trial A close correlation has been found, over a concentration for safety in normal healthy volunteers [35], AMD3100 range of 0.1–1000 ng/ml, between the AMD3100 con- recently entered phase II clinical trials in HIV-infected centrations required to inhibit (i) HIV-1 NL4-3 replication, individuals. (ii) monoclonal antibody (mAb 12G5) binding to the Given their high potency and selectivity as CXCR4 CXCR4 coreceptor, and (iii) SDF-1-induced signal trans- antagonists, bicyclams, such as AMD3100, may not only duction (Ca2+ flux), suggesting an intimate relationship have great potential for the therapy and/or prophylaxis of between these three parameters [29,30]. The inhibitory X4 HIV infections, but also other pathologic processes, effects of AMD3100 on the T-tropic HIV-1 NL4-3 strain such as breast cancer metastasis, which are at least partially have been demonstrated in a wide variety of cells expressing dependent of, or mediated by, signaling through CXCR4 CXCR4, including PBMCs; and, vice versa, various T- [36]. tropic and dual-tropic, but not M-tropic, HIV-1 strains have proven sensitive to AMD3100 in PBMC. Negatively charged amino acid (i.e. aspartic acid) resi- 4. Viral fusion (gp41) inhibitors dues in the extracellular regions of CXCR4 must be involved in its interaction with both AMD3100 and SDF- The interaction of the X4 or R5 HIV-1 envelope gly- 1, and the V3 loop of X4 HIV gp120, which are all three coprotein gp120 with the coreceptor CXCR4 or CCR5, highly basic. Substitutions of a neutral amino acid residue respectively, is followed by a spring-loaded action of the for aspartic acid in the second extracellular loop generated viral glycoprotein gp41 (normally covered by the bulkier resistance to AMD3100 [31]. In particular, the aspartate gp120), which then anchors through its amino terminus residues at positions 171 and 262, located close to the (the ‘‘fusion peptides’’) into the target cell membrane. This extracellular sides of the transmembrane segments TM4 initiates the fusion of the two lipid bilayers, that of the 262 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 viral envelope with that of the cellular plasma membrane The betulinic acid derivative RPR 103611 (5) is the [37]. At the onset of the fusion process, the hydropho- only nonpeptidic low-molecular-weight compound that has bic grooves on the surface of the N36 coiled coil in the been reported to block HIV-1 infection through interaction gp41 ectodomain become available for binding with ex- with gp41: this triterpene derivative has been found to traneous inhibitors, such as DP-178 (T-20), a 36-residue inhibit the infectivity of a number of HIV-1 strains in the peptide, that binds to the hydrophobic groove of N36 10 nM concentration range [41], apparently through inter- [37]. ference with a post-binding, envelope-dependent step T-20 (pentafuside) (4) is a synthetic, 36-amino acid involved in the fusion of the virus with the cell plasma peptide corresponding to residues 127–162 of the ectodo- membrane. main of gp41 (or residues 643–678 in the gp160 pre- The exact mode of action of RPR 103611 remains to be cursor). T-20, previously called DP-178, was modeled elucidated. Sequence analysis of RPR103611-resistant mu- after a specific domain (within gp41) predictive of a- tants indicated that a single amino acid change, I84S, in helical secondary structure: DP-178 consistently afforded HIV-1 gp41 is sufficient to confer drug resistance [42]. 100% blockade of virus-mediated cell–cell fusion (syncy- However, this I84S mutation did not occur in some of the tium formation) at concentrations ranging from 1 to 10 ng/ naturally RPR103611-resistant HIV-1 strains such as NDK. ml, i.e. 104-to105-fold lower than the cytotoxic concen- More recently, the action of RPR103611 has been thought to tration [38]. An initial clinical trial has been carried out depend on the accessibility of gp41 [43], and for the with T-20 at four doses (3, 10, 30 and 100 mg twice daily, isomeric betulinic acid derivative IC 9564, HIV-1 gp120, intravenously, for 14 days) in 16 HIV-infected adults: at rather than gp41, has been proposed as the prime target the highest dose (100 mg, twice daily), T-20 achieved by (based on the mutations G237R and R252K emerging in the 15th day a 1.5- to 2.0-fold reduction in plasma HIV gp120 of drug-resistant mutants) [44]. YK-FH312, a betu- RNA [39]. These data provide proof-of-concept that HIV linic acid derivative unrelated to RPR103611 or IC 9564, fusion inhibitors are able to reduce virus replication in was reported to block the assembly and/or budding of HIV vivo. particles [45].

Meanwhile, T-20 has proceeded to phase II/III clinical 5. Nucleocapsid protein (NCp7) Zn finger-targeted trials, and phase I clinical trials have been initiated with T- agents 1249, a 39-amino acid peptide derived from DP-107 (which is a 38-amino acid peptide corresponding to residues 558– The two zinc fingers [Cys-X2-Cys-X4-His-X4-Cys 595 of gp160); T-1249 would be 10-fold more potent than (CCHC), whereby X = any amino acid] in the nucleocapsid T-20 when evaluated in vitro against HIV under the same (NCp7) protein [46] comprise the proposed molecular target conditions [40]. for zinc-ejecting compounds such as 3-nitrosobenzamide E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 263

(NOBA), 2,2V-dithiobisbenzamide (DIBA), SRR-SB3 already equipped with a phosphonate group, and therefore (cyclic DIBA) [47], 1,2-dithiane-4,5-diol,1,1-dioxide (di- only need two phosphorylation steps to be converted to the thiane) [48] and azadicarbonamide (ADA) [49,50]. These active metabolite [58]. From PMEA and PMPA, the oral compounds should be able to interfere with both early prodrug forms [bis(pivaloyloxymethyl)-9-(2-phosphonylme- (uncoating, disassembly) and late phases (packaging, assem- thoxyethyl)adenine (bis(POM)-PMEA) or adefovir dipi- bly) of retrovirus replication. Their effect at the early phase voxyl (7), and bis(isopropyloxycarbonyloxymethyl)-(R)-9- (disassembly) may also be ascribed to cross-linkage among (2-phosphonylmethoxypropyl)adenine (bis(POC)-PMPA) or adjacent zinc fingers. The DIBAs are able to enter intact tenofovir disoproxil (8) fumarate, respectively] have been virions and the cross-linkage of NCp7 in virions correlates prepared. The former is in advanced phase III clinical trials with loss of infectivity and decreased proviral DNA syn- for the treatment of hepatitis B virus (HBV) infections, thesis during acute infection [51]. Electron microscopically, whereas the latter has completed phase III clinical trials for the effect bestowed by DIBAs on virus morphology could the treatment of HIV infections. A new drug application be described as ‘‘core-freezing’’ [52]. (NDA) and market authorization application (MAA) has been recently filed for tenofovir disoproxil fumarate with the FDA (US) and EMEA (EU), respectively. In rhesus macaques infected with the highly pathogenic chimeric virus SHIV, tenofovir treatment initiated 1 week post-infection, at a time when disseminated infection and extensive viral replication had already been established and CD4 + T-cell loss had begun, led to prompt, virtually complete suppres- sion of viral replication and long-term stabilization of CD4 + T-cell levels, which were sustained, even after withdrawal of Although NOBA, DIBA, dithiane and ADA have been tenofovir (after 12 weeks of treatment) [59]. shown to dock nicely on the NCp7 Zn finger domains [53] and are believed to selectively target these Zn fingers without affecting the cellular Zn finger proteins, their selectivity indexes [ratio of CC50 (50% cytotoxic concen- tration) over EC50 (50% effective concentration)] are not that impressive [53]. Of the NCp7-targeted compounds, ADA (6) has been the first to proceed to phase I/II clinical trials in advanced AIDS patients. Some preliminary evi- dence of efficacy was witnessed with add-on ADA in patients failing current antiretroviral therapy [54]; these studies should be further extended. Although ADA is an HIV NCp7 Zn-finger inhibitor, its action in vivo is likely to be multipronged. ADA may well interact with a variety of targets and, certainly, its inhibitory effects on T-cell responses in vitro and in vivo [55] can hardly be attributed to an action targeted at the HIV NCp7 Zn fingers.

6. RT inhibitors targeted at the substrate binding site

The substrate (dNTP) binding site of the HIV-1 RT is the target for a large variety of NRTI analogues, which have for several years [56] been recognized as efficacious drugs for the treatment of HIV infections: i.e. AZT, ddI, ddC, d4T, 3TC, ABC, and the yet experimental drug emtricitabine [( À )FTC]. Fozivudine tidoxil is a thioether lipid AZT conjugate that has recently passed phase II clinical trials [57] and should be as effective as, and potentially better tolerated than, AZT. As a rule, all these compounds must be phosphorylated to their 5V-triphosphate form, before they can act as competitive inhibitors/substrate analogues/chain ter- minators at the RT level. In contrast to the nucleoside analogues, the nucleotide analogues PMEA and PMPA are 264 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275

In addition to 3TC and ( À )FTC, the structurally related ( F )2V-deoxy-3V-oxa-4V-thiocytidine (BCH-10652,dOTC) [60], the dioxolane purine nucleoside analogues [61], the methylenecyclopropane nucleoside analogues (and their phosphoro-L-alaninate diesters) [62,63] and the 4V-ethynyl nucleoside analogues [64] have recently been described as new anti-HIV agents. [( À )FTC] (9) is in phase III trials for HIV and phase I/II trials for HBV; it is considered for use in the multidrug combination therapy of HIV-1 and HBV infections. Amdoxovir [DAPD, ( À )-h-D-2,6-diaminopur- ine dioxolane] (10), which is converted by adenosine deaminase to dioxolane guanine (DXG), has proven active against AZT- and 3TC-resistant HIV-1 strains and has proceeded to phase I/II clinical studies [65,66]. BCH- 10652 (dOTC) (11) has demonstrated activity against HIV-1 in the SCID-hu Thy/Liv model. Despite its struc- tural similarity to 3TC, dOTC proved also active against 3TC-resistant HIV-1 (M184V), albeit at a relatively high dosage level (400 mg/kg/day) [67]. Also in vitro, dOTC and its (+) and ( À ) enantiomers still retained, albeit reduced, activity against 3TC-resistant M184V and M184I HIV-1 mutants [68].

phosphate moiety a phenyl group and the methylester of alanine linked to the phosphate group through a phos- phoramidate linkage] have been constructed [70–72]. After the d4T aryloxyphosphoramidate (13) has been taken up by the cells, d4TMP is released intracellularly The bottleneck in the metabolic pathway leading from and then processed onto its active metabolite d4TTP AZT and the other 2V,3V-dideoxynucleoside (ddN) ana- [73]. This ‘‘thymidine kinase bypass’’ explains the high logues to their active 5V-triphosphate form is the first anti-HIV activity of d4T aryloxyphosphoramidate deriva- phosphorylation step. Therefore, attempts have been made tives in thymidine kinase deficient cells and resting mono- at constructing 2V,3V-dideoxynucleotide (ddNMP) prodrugs cytes/macrophages [74]. The thymidine kinase (in the that, once they have been taken up by the cells, deliver case of d4T) and the adenosine deaminase (in the case the nucleotide (ddNMP) form. This approach has proven of ddA) can also be bypassed by using the cyclic sali- particularly successful for a number of NRTIs such as genyl approach [75,76]. Cyclosaligenyl pronucleotides of 2V,3V-dideoxyadenosine (ddA) and d4T. Thus, the bis(S- d4T and ddA deliver exclusively the nucleotides d4TMP acetyl-2-thioethyl)phosphotriester of ddA [bis(SATE]d- and ddAMP, not only under chemical-simulated hydrol- dAMP] (12) was synthesized and found to be 1000-fold ysis conditions but also under intracellular conditions more potent against HIV than the parent compound ddA [77,78]. This has been convincingly shown for the cyclo- [69]. Similarly, aryloxyphosphoramidate derivatives of saligenyl derivative of d4TMP (14) in a number of cell d4T [i.e. So324, a d4T-MP prodrug containing at the lines [79]. E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 265

7. RT inhibitors targeted at the allosteric, nonsubstrate treatment of HIV-1 infections, emivirine (MKC-442) (15) binding site is in advanced (phase III) clinical trials, and others are in preclinical or early clinical development. The NNRTIs More than 30 structurally different classes of com- interact with a specific ‘‘pocket’’ site of the HIV-1 RT pounds have been identified as NNRTIs, viz. compounds [81], which is closely associated with, but distinct from, that are specifically inhibitory to HIV-1 replication and the substrate binding site. NNRTIs are notorious for targeted at a nonsubstrate binding site of the RT [80]. rapidly eliciting resistance [82], resulting from mutations Three NNRTIs (nevirapine, delavirdine and efavirenz) at the amino acid residues that surround the NNRTI-bind- have so far been formally licensed for clinical use in the ing site of HIV-1 RT. However, emergence of NNRTI- 266 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 resistant HIV strains can be prevented if the NNRTIs are urea-PETT derivatives [97], the alkenyldiarylmethane combined with NRTIs and used from the beginning at (ADAM) series of compounds [98], the pyrrolobenzoxaze- sufficiently high concentrations [80]. pinone (PBO) derivatives [99], the quinoxalinylethylpyridyl The thiocarboxanilide UC-781 (16) is an exceptionally thioureas (QXPTs) [100], the emivirine (MKC-442) deriv- potent inhibitor of HIV-1 replication [80]. It has been found ative SJ-3366 [101] and R165335(TMC125) [102]. As a to restore the antiviral activity of AZT against AZT-resistant rule, the ‘‘new’’ (‘‘second’’ or ‘‘third’’ generation) NNRTIs HIV-1 [83]. UC-781 has been recognized as a (retro)viru- exhibit higher potency than the ‘‘old’’ (‘‘first’’ generation) cidal agent, capable of reducing the infectivity of HIV-1 NNRTIs against wild-type and NNRTI-resistant HIV-1 virions, and, therefore, yielding considerable promise for the [91,94–96,99,102]. This is particularly prominent for use in (retro)virucidal formulations to prevent the trans- DPC 083 (17) and R165335 (TMC125) (18) that showed mission of HIV from infected to noninfected individuals activity against L100I, K103N, Y181C, Y188L, [84]. UC-781 would seem an ideal candidate for application K103N + L100I and K103N + Y181C RT mutant strains in as a vaginal microbicide (virucide), i.e. when formulated in the nanomolar concentration range [102]. This makes replens gel [85]. R165335 (TMC125) an excellent candidate for further clinical development.

To the new classes of NNRTIs that offer potent anti-HIV- 1 activity belong the thieno[3,4][1,2,4]thiadiazine derivative QM96521 [86], the quinoxaline GW420867X [87], the imidazole derivative S-1153 (AG1549, ) [88– 90], ( À )-6-chloro-2-[(1-furo[2,3-c]pyridin-5-yl-ethyl)thio]- 4-pyrimidinamine (PNU-142721) [91], N-[2-(2,5-dimethox- yphenylethyl]-NV-[2-(5-bromopyridyl]-thiourea (HI-236) [92], the pyrido[1,2a]indole derivative BCH-1 [93], the 4- Some of the new NNRTIs, such as SJ-3366 (19), possess cyclopropylalkynyl-4-trifluoromethyl-3,4-dihydro-2(1H) remarkable features. This compound was reported to inhibit quinazolinones DPC 961 and DPC 963, the 4-cyclopropylal- HIV-1 replication at a concentration below 1 nM with a kenyl-4-trifluoromethyl-3,4-dihydro-2(1H)quinazolinones therapeutic index greater than 4,000,000, and to inhibit HIV- DPC 082 and DPC 083 [94], the thiophene-ethylthiourea 2 replication (albeit at higher concentrations than those (TET) derivative HI-443 [95], the cyclohexenylethylthiourea required for inhibition of HIV-1) at the viral entry stage derivatives HI-346 and HI-445 [96], the cis-cyclopropyl [101]. E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 267

(+)- (22) is the only naturally occurring NNRTI: it was first isolated from a tropical tree (Calophyl- llum lanigerum) and has already been the subject of a phase I clinical study in healthy, HIV-negative individuals [103].

Capravirine (AG1549) (20) has a favorable profile of resilience to many drug resistance mutations, which has been attributed to extensive main chain hydrogen bonding involv- ing the main chain of residues 101, 103, and 236 of the p66 RT subunit [89]. Capravirine has proceeded to phase II/III clinical trials [90].

Recently, an unexpected effect of NNRTIs on HIV-1 RT dimerization was documented [104]: several NNRTIs, including efavirenz, were found to enhance the association between the RT subunits p66 and p51, apparently due to a conformational change in the p66 subunit that resulted in enhanced binding to the p51 subunit. It remains to be established if this enhanced dimerization has any bearing on the anti-HIV-1 potency of the NNRTIs.

8. HIV integrase inhibitors

Retrovirus integration requires at least two viral com- ponents, the retroviral enzyme integrase, and cis-acting sequences at the retroviral DNA termini U3 and U5 ends The NNRTIs cis-cyclopropylurea-PETT [97] and PBO of the long terminal repeats (LTRs). Since HIV, like other derivatives [99] are orally bioavailable and penetrate well retroviruses, cannot replicate without integration into a into the brain. The broad, potent antiviral activity, and host chromosome, integrase has been considered as an favorable pharmacokinetic profile, have led to the selection attractive therapeutic target. Numerous compounds have of PNU-142721 (21) for clinical studies [91]; and DPC 961, been described as inhibitors of HIV-1 integrase (for a DPC 963, DPC 082 and DPC 083 for clinical development recent review, see Ref. [105]): for example, polyamides, [94]. bisdistamycins and lexitropsins [106], polyhydroxylated aromatic type of compounds, including ellagic acid, pur- purogallin, 4,8,12-trioxatricornan and hypericin [107] and a series of thiazolothiazepine derivatives, preferably pos- sessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups [108]. The problem with integrase inhib- itors is that while they might be effective in an enzyme- based assay, their anti-HIV activity in cell culture may be masked by cytotoxicity, and if they do exhibit anti-HIV activity, this could, at least in some cases, be attributed to antiviral actions targeted at other steps in the HIV repli- cative cycle. L-chicoric acid [109–111] is such a case. L-chicoric acid is structurally reminiscent of curcumin [112], 3,5-dicaf- 268 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 feoylquinic acid [113], rosmarinic acid [114] and dicaf- tives have been reported as inhibitors of HIV-1 integrase feoyltartaric acids (DCTAs) [115], and all these compounds [119]. have been reported to inhibit HIV-1 integrase. Integrase was identified as the molecular target for the action of L-chicoric acid (23) since a single amino acid substitution (G140S) in the integrase rendered the corresponding HIV-1 mutant resistant to L-chicoric acid [111]. We have recently demon- strated [116], however, that L-chicoric acid owes its anti- HIV activity in cell-culture to an interaction with the viral envelope gp120. Upon repeated passages of the virus in the presence of the compound, mutations were found in the V2, V3 and V4 loop of gp120, while no mutations were seen in the integrase. We did confirm that in an enzymatic assay L- chicoric acid inhibited HIV integrase activity, but integrase carrying the G140S mutation appeared to be as sensitive to the inhibitory effect of L-chicoric acid as the wild-type 9. Transcription (transactivation) inhibitors integrase. Furthermore, L-chicoric acid proved inactive against HIV strains that were resistant to polyanionic At the transcription level, HIV gene expression may be compounds known to interact at the virus adsorption level, inhibited by compounds that interact with cellular factors and time-of-addition experiments further corroborated an that bind to the LTR promoter and that are needed for basal interaction of L-chicoric acid at the virus adsorption stage level transcription, such as the NF-nB inhibitors [120]. [116]. Greater specificity, however, can be expected from those

Recently, the structure of the HIV-1 integrase core compounds that specifically inhibit the transactivation of the domain complexed with an inhibitor [1-(5-chloroindol-3- HIV LTR promoter by the viral Tat (trans-activating) yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone] has been protein [120]. Tat has pleiotropic effects: it not only acti- described as a platform for structure-based design of novel vates the transcription of HIV-1 RNA, but also binds to a HIV-1 integrase inhibitors [117]. This was followed by the number of receptors, i.e. on smooth muscle and skeletal description of a number of diketo acids (such as L-731,988 muscle cells [121]: the basic domain of Tat may be and L-708,906) as inhibitors of the integrase-mediated important, not only for translocation but also for nuclear strand transfer reaction that leads to the covalent linkage localisation and trans-activation, and thus targeting of the of the viral DNA 3Vends to the cellular (target) DNA [107]. Tat basic domain may provide great scope for therapeutic These compounds were also found to inhibit HIV-1 repli- intervention in HIV-1 infection [121]. cation in cell culture. Furthermore, mutations in the HIV-1 A number of compounds have been reported to inhibit integrase conferred resistance to the inhibitory effects of HIV-1 replication in both acutely and chronically infected the compounds on both strand transfer and HIV-1 infec- cells through interference with the transcription process: i.e. tivity [118]. Thus, it was surmised that these diketo acids fluoroquinoline derivatives [122]. The inhibitory effects of owe their antiviral activity exclusively to inhibition of one the fluoroquinolines (K-12) [8-difluoromethoxy-1-ethyl-6- of the two catalytic functions of integrase, namely strand fluoro-1,4-dihydro-7-[4-(2-methoxyphe-nyl)-1-piperazinyl]- transfer [the other catalytic function being endonucleolytic 4-oxoquinoline-3-carboxylic acid] and (K-37) [7-(3,4-dehy- processing of the (pro)viral DNA to remove the terminal dro-4-phenyl-1-piperidinyl)-1,4-dihydro-6-fluoro-1-methyl- dinucleotide (GT) from the 3Vend]. Starting from L-731, 8-trifluoromethyl-4-oxoquinoline-3-carboxylic acid] (25)on 988 (24), additional 4-aryl-2,4-dioxobutanoic acid deriva- the HIV-1 LTR-driven gene expression may at least in part E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 269 be attributed to inhibition of Tat [123] or other RNA- replication in both acutely and chronically infected cells. dependent transactivators [124]. Resistance was generated upon serial passage of the virus in the presence of temacrazine and was associated with several unique nucleotide changes in HIV-1 LTR at positions À 1, À 2 and + 111 relative to the start of transcription [126]. Tat peptide analogs, encompassing the Tat core domain (amino acid residues 36–50) [127], or the basic domain (amino acids 48–56: RKKRRQRRR) [128] have been reported to inhibit HIV-1 replication, and, as expected, these peptide analogs were able to effectively block the Tat transactivation process. The 9-mer peptoid CGP64222 (27), which is structurally reminiscent of the amino acid 48–56 sequence RKKRRQRRR of Tat, was also reported, on the one hand, to block the Tat/TAR interaction, and, on the other hand, to suppress HIV-1 replication [129]. We The bistriazoloacridone temacrazine [1,4-bis(3-(6-oxo- have demonstrated, however, that the peptoid CGP64222 6H-v-triazolo[4,5,1]acridin-5-yl-aminopropyl)piperazine] owes its anti-HIV activity in cell culture primarily to an (26) was found to block HIV-1 RNA transcription from the interaction with CXCR4, the coreceptor for X4 HIV strains HIV proviral DNA without interfering with the transcription [130], which is, perhaps, not surprising given the structural of any cellular genes [125]: the compound inhibited HIV-1 similarity of CGP64222 to the other, polypeptidic, CXCR4 270 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 antagonists such as T22 [23] and nona-arginine (ALX40- 10. HIV PIs 4C) [24]. In fact, Tat itself (following its extracellular release) has recently been shown to block CXCR4-depend- HIV PIs prevent the cleavage of the gag and gag-pol ent HIV-1 infection [131], presumably through blockade of precursor polyproteins to the structural proteins (p17, p24, CXCR4 by the above-mentioned 48–56 amino acid portion p7, p6, p2, p1) and functional proteins (protease, RT/RNase (RKKRRQRRR) of the molecule. H, integrase), thus arresting maturation and thereby block- Flavopiridol (L86-8275, HMR1275) is a cyclin-depend- ing infectivity of the nascent virions [133]. The HIV PIs ent kinase (Cdk) and P-TEFb inhibitor, which is in clinical have been tailored after the target peptidic linkage in the gag trials for the treatment of cancer because of its antiprolifer- and gag-pol polyproteins that are cleaved by the protease, ative properties. P-TEFb is a protein kinase composed of viz. the phenylalanine–proline sequence at positions 167 Cdk9 and cyclin T1 and secures the elongation phase of and 168 of the gag-pol polyprotein. All PIs that are currently transcription by RNA polymerase II (through phosphoryla- licensed for the treatment of HIV infection, namely saqui- tion of the carboxyl-terminal domain). Tat forms a triple navir, ritonavir, indinavir, nelfinavir, amprenavir and lopi- complex with P-TEFb (composed of Cdk9 and cyclin T1) navir, share the same structural determinant, i.e. an and the nascent transcript from the HIV-1 LTR promoter. hydroxyethylene (instead of the normal peptidic) bond, Consistent with its ability to block P-TEFb, flavopiridol (28) which makes them nonscissile substrate analogues for the was found to block Tat transactivation, and, concomitantly, HIV protease. All six licensed PIs follow the same principle; also inhibited HIV replication [132]. that is, they act as peptidomimetic inhibitors of HIV protease [134]. Lopinavir is co-dosed with ritonavir at 400/100 mg twice daily. The reason for this combination is that ritonavir strongly inhibits the metabolism of lopinavir and allows lopinavir to reach much higher plasma drug levels upon oral administration [135]. In phase III clinical trials is (BMS-232632) (29), which has been accredited with a favorable resistance profile that does not parallel any of the other PIs currently in clinical use, as well as a favorable pharmacokinetic profile that would allow once-daily dosing [136]. Resistance mutations have been reported for most, if not all, peptidomimetric inhibitors of HIV protease. This has prompted the search for new, nonpeptidic inhibitors of HIV protease, which, in addition to a broader anti-HIV activity spectrum, might also offer increased oral bioavailability and/or pharmacokinetic properties. Examples of nonpeptidic PIs of HIV protease include 4-hydroxycoumarins and 4- E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 271 hydroxy-2-pyrones [137], sulfonamide-substituted deriva- shown to inhibit gag polyprotein processing as well as tives [138], cyclic ureas (i.e. DMP-323 and DMP-450) HIV maturation and release [148]. While a potentially [139,140], cyclic cyanoguanidines [141], aza-dipeptide ana- interesting approach, it remains to be seen whether inhib- logues [142], and tipranavir (PNU-140690), a sulfonamide- itors of the proteasome/ubiquitin system display sufficient containing 5,6-dihydro-4-hydroxy-2-pyrone [143–145]. specificity in their anti-HIV action so as to suppress virus The major advantage of the cyclic urea mozenavir (DMP- replication without (overt) cytotoxicity. 450) (30) is its substantial oral bioavailability observed in all species examined, including man [140]. DMP-450 has been the subject of phase I/II dose-escalating clinical studies and 11. Conclusions appears to have good antiviral activity and tolerability at all doses tested [146]. The new aza-dipeptide analogues com- In recent years, an ever increasing number of compounds bine excellent anti-HIV potency with high blood drug levels have been uncovered as anti-HIV agents targeted at virtually after oral administration, and, furthermore, they show no any step of the virus replicative cycle: adsorption, entry, cross-resistance with saquinavir-resistant HIV strains [142]. fusion, uncoating, reverse transcription, integration, tran- Tipranavir (31) showed low cross-resistance to HIV strains scription (transactivation) and maturation. In addition to the that were resistant to the established (peptidomimetic) ‘‘newer’’ NRTIs, NNRTIs and PIs, various other com- inhibitors of HIV protease [145]. Also, tipranavir retained pounds, i.e. those that are targeted at viral entry (i.e. CXCR4 marked activity against HIV-1 isolates derived from patients and CCR5 antagonists) and virus-cell adsorption/fusion (i.e. with multidrug resistance to other PIs [147]. compounds interacting with either gp120 or gp41), offer

Independently of the HIV protease itself, proteasomes great potential for the treatment of HIV infections. Quite a play a role in the processing of the gag polyprotein, and number of compounds are capable of interacting with more proteasome inhibitors, such as epoxomicin, have been than one target. Two examples in point are the DCTA L- 272 E. De Clercq / Biochimica et Biophysica Acta 1587 (2002) 258–275 chicoric acid, and the nonapeptoid CGP 64222. L-chicoric [18] T. Dragic, A. Trkola, D.A. Thompson, E.G. Cormier, F.A. Kajumo, acid was originally identified as an integrase inhibitor, and E. Maxwell, S.W. Lin, W. Ying, S.O. Smith, T.P. Sakmar, J.P. Moore, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 5639–5644. the nonapeptoid as a transactivation (Tat) antagonist, and [19] M. Shiraishi, Y. Aramaki, M. Seto, H. Imoto, Y. Nishikawa, N. Kan- their anti-HIV activity in acutely infected cells was ascribed zaki, M. Okamoto, H. Sawada, O. Nishimura, M. Baba, M. 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